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RNA-mediated interaction of Cajal bodies and U2 snRNA genes.

Frey MR, Matera AG - J. Cell Biol. (2001)

Bottom Line: Although promoter substitution arrays colocalized with CBs, constructs containing intragenic deletions did not.Together, the results illustrate a specific requirement for both the snRNA transcripts as well as the presence of snRNPs (or snRNP proteins) within CBs.Our data thus provide significant insight into the mechanism of CB interaction with snRNA loci, strengthening the putative role for this nuclear suborganelle in snRNP biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Program in Cell Biology, Case Western Reserve University, Cleveland, OH 44106, USA.

ABSTRACT
Cajal bodies (CBs) are nuclear structures involved in RNA metabolism that accumulate high concentrations of small nuclear ribonucleoproteins (snRNPs). Notably, CBs preferentially associate with specific genomic loci in interphase human cells, including several snRNA and histone gene clusters. To uncover functional elements involved in the interaction of genes and CBs, we analyzed the expression and subcellular localization of stably transfected artificial arrays of U2 snRNA genes. Although promoter substitution arrays colocalized with CBs, constructs containing intragenic deletions did not. Additional experiments identified factors within CBs that are important for association with the native U2 genes. Inhibition of nuclear export or targeted degradation of U2 snRNPs caused a marked decrease in the levels of U2 snRNA in CBs and strongly disrupted the interaction with U2 genes. Together, the results illustrate a specific requirement for both the snRNA transcripts as well as the presence of snRNPs (or snRNP proteins) within CBs. Our data thus provide significant insight into the mechanism of CB interaction with snRNA loci, strengthening the putative role for this nuclear suborganelle in snRNP biogenesis.

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SMN gems, which lack snRNPs, do not colocalize with RNU2 loci. For better separation of gems and CBs, HeLa-PV cells were grown at 32°C before fixation and staining with anticoilin (top, red) and anti-SMN (bottom, white) antibodies. Subsequently, DNA FISH was performed with an RNU2 probe to detect U2 genes (green signals, both panels). Of the ∼100 cells examined, RNU2 loci were never observed to overlap with non-snRNP–containing SMN gems. Arrows mark a RNU2–CB association (top) that does not colocalize with a gem (bottom).
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fig5: SMN gems, which lack snRNPs, do not colocalize with RNU2 loci. For better separation of gems and CBs, HeLa-PV cells were grown at 32°C before fixation and staining with anticoilin (top, red) and anti-SMN (bottom, white) antibodies. Subsequently, DNA FISH was performed with an RNU2 probe to detect U2 genes (green signals, both panels). Of the ∼100 cells examined, RNU2 loci were never observed to overlap with non-snRNP–containing SMN gems. Arrows mark a RNU2–CB association (top) that does not colocalize with a gem (bottom).

Mentions: There are obviously two sides to the RNU2–CB interaction. Thus far, we have concentrated on the chromosomal side of the association. Are there also certain requirements for components on the CB side? We hypothesized that snRNPs returning from the cytoplasm and accumulating in CBs could provide the cell with an opportunity for autogenous feedback regulation (Frey and Matera, 1995; Matera, 1998; Frey et al., 1999). Thus, it seemed possible that the presence of (partially) mature snRNPs within the CB might be necessary for association with snRNA genes. We therefore analyzed a strain of HeLa cells, called HeLa-PV, in which SMN gems and CBs are often observed as separate structures (Liu and Dreyfuss, 1996; Matera and Frey, 1998; Sleeman and Lamond, 1999b). In those instances when CBs and gems occupy distinct locations, snRNPs invariably reside in the coilin-positive CBs and are not detectable in SMN gems. Growing the HeLa-PV cells at 32°C can exacerbate the separation phenotype (Liu and Dreyfuss, 1996). In this case, we wanted to determine whether U2 genes associate with snRNP-positive CBs or snRNP-negative SMN gems. To this end, we differentially labeled CBs and gems and subsequently hybridized a DNA probe to RNU2 loci. Of all cells examined (n > 100), not one exhibited an RNU2 array colocalized with a gem (Fig. 5) . Thus U2 genes always colocalized with snRNP- and coilin-positive CBs.


RNA-mediated interaction of Cajal bodies and U2 snRNA genes.

Frey MR, Matera AG - J. Cell Biol. (2001)

SMN gems, which lack snRNPs, do not colocalize with RNU2 loci. For better separation of gems and CBs, HeLa-PV cells were grown at 32°C before fixation and staining with anticoilin (top, red) and anti-SMN (bottom, white) antibodies. Subsequently, DNA FISH was performed with an RNU2 probe to detect U2 genes (green signals, both panels). Of the ∼100 cells examined, RNU2 loci were never observed to overlap with non-snRNP–containing SMN gems. Arrows mark a RNU2–CB association (top) that does not colocalize with a gem (bottom).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196410&req=5

fig5: SMN gems, which lack snRNPs, do not colocalize with RNU2 loci. For better separation of gems and CBs, HeLa-PV cells were grown at 32°C before fixation and staining with anticoilin (top, red) and anti-SMN (bottom, white) antibodies. Subsequently, DNA FISH was performed with an RNU2 probe to detect U2 genes (green signals, both panels). Of the ∼100 cells examined, RNU2 loci were never observed to overlap with non-snRNP–containing SMN gems. Arrows mark a RNU2–CB association (top) that does not colocalize with a gem (bottom).
Mentions: There are obviously two sides to the RNU2–CB interaction. Thus far, we have concentrated on the chromosomal side of the association. Are there also certain requirements for components on the CB side? We hypothesized that snRNPs returning from the cytoplasm and accumulating in CBs could provide the cell with an opportunity for autogenous feedback regulation (Frey and Matera, 1995; Matera, 1998; Frey et al., 1999). Thus, it seemed possible that the presence of (partially) mature snRNPs within the CB might be necessary for association with snRNA genes. We therefore analyzed a strain of HeLa cells, called HeLa-PV, in which SMN gems and CBs are often observed as separate structures (Liu and Dreyfuss, 1996; Matera and Frey, 1998; Sleeman and Lamond, 1999b). In those instances when CBs and gems occupy distinct locations, snRNPs invariably reside in the coilin-positive CBs and are not detectable in SMN gems. Growing the HeLa-PV cells at 32°C can exacerbate the separation phenotype (Liu and Dreyfuss, 1996). In this case, we wanted to determine whether U2 genes associate with snRNP-positive CBs or snRNP-negative SMN gems. To this end, we differentially labeled CBs and gems and subsequently hybridized a DNA probe to RNU2 loci. Of all cells examined (n > 100), not one exhibited an RNU2 array colocalized with a gem (Fig. 5) . Thus U2 genes always colocalized with snRNP- and coilin-positive CBs.

Bottom Line: Although promoter substitution arrays colocalized with CBs, constructs containing intragenic deletions did not.Together, the results illustrate a specific requirement for both the snRNA transcripts as well as the presence of snRNPs (or snRNP proteins) within CBs.Our data thus provide significant insight into the mechanism of CB interaction with snRNA loci, strengthening the putative role for this nuclear suborganelle in snRNP biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Program in Cell Biology, Case Western Reserve University, Cleveland, OH 44106, USA.

ABSTRACT
Cajal bodies (CBs) are nuclear structures involved in RNA metabolism that accumulate high concentrations of small nuclear ribonucleoproteins (snRNPs). Notably, CBs preferentially associate with specific genomic loci in interphase human cells, including several snRNA and histone gene clusters. To uncover functional elements involved in the interaction of genes and CBs, we analyzed the expression and subcellular localization of stably transfected artificial arrays of U2 snRNA genes. Although promoter substitution arrays colocalized with CBs, constructs containing intragenic deletions did not. Additional experiments identified factors within CBs that are important for association with the native U2 genes. Inhibition of nuclear export or targeted degradation of U2 snRNPs caused a marked decrease in the levels of U2 snRNA in CBs and strongly disrupted the interaction with U2 genes. Together, the results illustrate a specific requirement for both the snRNA transcripts as well as the presence of snRNPs (or snRNP proteins) within CBs. Our data thus provide significant insight into the mechanism of CB interaction with snRNA loci, strengthening the putative role for this nuclear suborganelle in snRNP biogenesis.

Show MeSH
Related in: MedlinePlus